Method and composition for hyperthermally treating cells

ABSTRACT

A method for the hyperthermia treatment of tissue in a target site applies a heat source to kill cells without protein denaturization. The method introduces an encapsulated dye that is released at a selected temperature in the target site to indicate that a threshold temperature has been reached to hyperthermally treat the tissue. In one embodiment, the composition releases the dye at a temperature of 42° C. to 50° C., and preferably about 45° C. to 49° C. The composition which can be a liposome composition encapsulating the dye can be introduced to the bloodstream of the patient to flow through the target site.

FIELD OF THE INVENTION

[0001] The present invention relates to a method and composition forhyperthermally treating cells at a site in the body. More particularly,the present invention relates to a method for treating cells at a targetsite in the body, such as at a lens capsule of an eye, tumors, andexudative ARM (age related macular degeneration) by applying thermalenergy to the target site to heat the cells to a temperature which willkill the cells or impede cell multiplication without exceeding theprotein denaturation temperature of the tissue.

BACKGROUND OF THE INVENTION

[0002] Several techniques currently exist for treating cells at aselected site in the body with heat or chemicals to kill or impedemultiplication of those cells to prevent undesired cell proliferation.For example, numerous types of chemotherapy drugs exists which, wheninjected into a tumor or delivered systemically to a patient, attack andkill cancerous cells to prevent them from further multiplying.

[0003] Radiation techniques can also be used to kill cancerous or otherundesired cells. Cell death begins to occur when the cells are heated toa temperature of about 5° C. or more above the normal body temperatureof 37° C. Applying radiation to a localized site in the body, such as atumor or other area containing undesired cells, can heat the cells atthe site to temperatures in excess of 60° C. Such high temperaturescauses a phenomenon known as protein denaturation to occur in the cells,which results in immediate cell death. Accordingly, radiation therapyhas been suitable in successfully treating certain types of cancers andother diseases involving uncontrolled cell growth.

[0004] Other types of heating techniques, such as the use of probes orcatheters to provide localized heat to a site of interest also exist.Like radiation therapy, these techniques also heat the cells to atemperature sufficient to cause protein denaturation in the cells tothus kill the cells quickly.

[0005] Photosensitive chemicals are also used to kill cells at certainsites of interest in the body. For example, a photosensitive chemicalcan be injected directly into a site of interest to expose cells at thatsite to the chemical. A light emitting source, which emits light at awavelength that will activate the photosensitive chemical, is thenfocused on the site of interest. Accordingly, the light activates thephotosensitive chemical that has been absorbed by or is otherwisepresent in the cells of interest. The activated chemical kills thecells, which thus prevents undesired cell proliferation.

[0006] Although the techniques mentioned above can be suitable forpreventing certain types of cell proliferation and certain sites in thebody, several drawbacks with these techniques exist. For example, oftenthe use of chemotherapy drugs alone to treat a tumor or cancerous siteis insufficient to kill the undesired cells. Moreover, the chemotherapydrugs also kill many normal healthy cells along with the cancerouscells, which can adversely affect the patient's health.

[0007] The use of radiation in conjunction with chemotherapy can have amore detrimental effect on the cancerous cells. However, as withchemotherapy, radiation often kills normal healthy cells, such as thosein front of or behind the site of interest, along with the cancerouscells. Moreover, the intense heating of the cells can cause the cells tocoagulate and thus block the capillaries at the site of interest. Theblocked capillaries therefore prevent chemotherapy drugs from reachingthe site of interest.

[0008] One example of a method of chemically treating a target site isdisclosed in U.S. Pat. No. 6,248,727 to Zeimer. This method delivers aliposome containing a fluorescent dye and tissue-reactive agent. Theliposome is administered intravenously to flow to the locus in the eyeof the patient and the site is non-invasively heated to release the dyeand the tissue-reactive agent. The dye is fluoresced to observe thepattern of the fluorescence. The tissue-reactive agent is activated tochemically damage and occlude the blood vessel. The liposomes areselected to release the dye at a temperature of 41° C. or less withoutcausing thermal damage to the blood vessel.

[0009] In addition, the above techniques have not been used to preventunwanted cell proliferation at certain locations in the eye, such as atthe retina or at the lens capsule. Because the retina is very sensitive,conventional radiation techniques can be too severe to treat cancerouscells on, in or under the retina.

[0010] Also, after cataract surgery, a phenomenon known as capsularopacification and, in particular, posterior capsular opacification canoccur in which the epithelial cells on the lens capsule of the eyeexperience proliferated growth. This growth can result in the cellscovering all or a substantial portion of the front and rear surfaces ofthe lens capsule, which can cause the lens capsule to become cloudy andthus adversely affect the patient's vision. These cells can be removedby known techniques, such as by scraping away the epithelial cells.However, it is often difficult to remove all of the unwanted cells.Hence, after time, the unwanted cells typically will grow back, thusrequiring further surgery.

[0011] Accordingly, a need exists for a method for hyperthermallytreating tissue and preventing unwanted cell proliferation at sites inthe body, especially at sites in the eye such as the retina, choroid andlens capsule, which does not suffer from the drawbacks associated withthe known techniques discussed above.

SUMMARY OF THE INVENTION

[0012] The present invention is directed to a method of hyperthermallytreating tissue by heating the tissue above a temperature which killscells in the tissue. In particular, the invention is directed to amethod of heating tissue above a temperature effective to treat thetissue without denaturing the protein.

[0013] Accordingly, a primary aspect of the invention is to provide amethod for heating tissue at least to a temperature sufficient tohyperthermally treat the tissue.

[0014] Another aspect of the invention is to provide a method ofhyperthermally treating tissue to a temperature sufficient to kill cellsin the tissue and at a temperature below the protein denaturizationtemperature of the tissue.

[0015] A further aspect of the invention is to provide a method ofhyperthermally treating tissue, where the tissue includes or is providedwith a temperature indicator to indicate a hyperthermally effectivetemperature of the tissue.

[0016] Still another aspect of the invention is to provide a method ofhyperthermally treating tissue where a temperature indicator compositionis introduced into the tissue or bloodstream near the tissue to indicatea tissue temperature effective to hyperthermally treat the tissue and atemperature indicator to indicate a tissue temperature above a proteindenaturization temperature of said tissue.

[0017] A further aspect of the invention is to provide a method ofhyperthermally treating tissue by introducing a temperature indicatorinto the tissue and heating the tissue to a temperature where thetemperature indicator can be detected. In a preferred embodiment, thetemperature at which the indicator can be detected is a temperatureeffective to hyperthermally treat the tissue and is at a temperaturebelow the protein denaturization temperature.

[0018] A further aspect of the invention is to provide a method ofheating and detecting a temperature of a tissue between a firsttemperature and a second temperature. The method introduces atemperature indicator into the tissue. The temperature indicatorincludes a first dye that can be detected at the first temperature toindicate that the first temperature has been reached, and a second dyethat can be detected at the second temperature to indicate that thesecond temperature has been reached.

[0019] Still another aspect of the invention is to provide a temperatureindicating composition for introducing into a tissue to be thermallytreated. The composition includes a first dye encapsulated in a heatsensitive liposome where the first dye is releasable at a temperatureeffective to hyperthermally treat the tissue and at a temperature belowthe protein denaturization temperature. The composition also includes asecond dye encapsulated in a second liposome where the second dye isreleasable at a temperature at or above the protein denaturizationtemperature.

[0020] Another aspect of the invention is to provide a method tohyperthermally treat tissue to kill the tissue cells substantiallywithout protein denaturization of the tissue where the tissue includes aheat sensitive liposome containing a temperature indicating dye and atemperature activated bioactive compound. The tissue is heated torelease the dye from the liposome to indicate a thermally effectivetemperature to kill cells in the tissue at a temperature below theprotein denaturization temperature. The heat applied to the tissuesimultaneously releases the bioactive compound to treat the tissue.

[0021] The various aspects of the invention are basically attained byproviding a method of hyperthermally treating tissue in an animal. Themethod comprises the step of introducing a temperature indicatingsubstance into the bloodstream of the animal to flow through a targetsite. The temperature indicating substance includes a fluorescent dyeencapsulated within a heat sensitive liposome. The fluorescent dye isreleasable from the liposome at a temperature of at least 41° C. A heatsource is applied to the target site and the target is hyperthermallyheated to at least 41° C. to release and fluoresce the dye and tohyperthermally treat the target site for a time sufficient to kill cellsin the tissue.

[0022] The aspects of the invention are also attained by providing amethod of detecting a threshold temperature and of hyperthermallytreating tissue in an animal. The method comprises the step ofintroducing a first fluorescent dye encapsulated in a first heatsensitive liposome into the bloodstream of an animal in a location toflow through a target site in the animal. The first fluorescent dye isreleasable from the first heat sensitive liposome at a temperature of atleast 41° C. The target site is heated to a temperature to release thefirst fluorescent dye and the first fluorescent dye is fluoresced toindicate and visualize a tissue temperature of at least 41° C. Heatingof the target site is continued at a temperature of at least 41° C for atime sufficient to hyperthermally treat the tissue.

[0023] The aspects of the invention are further attained by providing amethod of hyperthermally treating tissue of an animal. The methodcomprises the step of introducing a temperature indicating substanceinto the bloodstream of the animal to flow through a target site. Thetemperature indicating substance includes a first fluorescent dyeencapsulated in a first temperature sensitive liposome. The firstfluorescent dye is releasable from the first liposome by heating to atemperature of at least 42° C. A second fluorescent dye encapsulated ina second temperature sensitive liposome is also included. The secondfluorescent dye is releasable from the second liposome by heating to atemperature of at least 50° C. The target site is heated to atemperature of at least 42° C. The first fluorescent dye is fluorescedto indicate an effective temperature for hyperthermally treating thetissue without releasing the second fluorescent dye from the secondliposomes.

[0024] These and other aspects of the invention will become apparent toone skilled in the art in view of the following detailed description ofthe invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The following is a brief description of the drawing, in which:

[0026] The FIGURE is a schematic diagram of one embodiment of theinvention showing a probe for hyperthermally treating tissue andvisualizing a dye in the target site.

DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention is directed to a method and composition forhyperthermally treating tissue. In particular, the invention is directedto as method for heating tissue above a temperature effective to killtissue cells or inhibit multiplication of cells below the proteindenaturization temperature of the tissue.

[0028] The method of the invention introduces a composition into thebloodstream of the body in a location to flow into or through a targetsite to be treated. A heat source is applied to the target site to heatthe tissue in the target site for a time sufficient to hyperthermallytreat the tissue and activate the composition. As used herein, the term“hyperthermal” refers to a temperature of the cell or tissue that killsor damages the cells without protein denaturization.

[0029] The composition contains a temperature indicator that is able toprovide a visual indication when a minimum or threshold temperature isattained that is sufficient to hyperthermally treat the tissue. It is afeature of the invention to provide a method of heating tissue in atarget site to a hyperthermally effective temperature and to provide avisual indication that a temperature of at least 41° C., and preferablyat least 42° C. is attained. In one embodiment, the composition includesa second temperature indicator to provide a visual indication when aprotein denaturization temperature is attained thereby providing anindication that a maximum desired temperature is exceeded. The heatsource can be applied to the tissue so that the composition provides anindication that a thermally effective temperature is attained that isbelow the protein denaturization temperature of the tissue.

[0030] In one embodiment of the invention, the method introduces acomposition to a target site, where the composition includes afluorescent dye that is encapsulated in a heat sensitive particle, suchas a liposome. The dye is a fluorescent dye that can be excited tofluoresce and be observed or visualized by the operator. Preferably, theheat sensitive liposomes are formed to rupture or release thefluorescent dye at a temperature at least equal to the temperaturenecessary to kill cells in the tissue and at a temperature below theprotein denaturization temperature. The composition containing the heatsensitive liposomes encapsulating the fluorescent dye is introduced intothe bloodstream to flow to or through the target site. The amount of theliposome composition is introduced in an amount effective to be releasedin or near the target site and to excited and visualized by the excitinglight source and the visualizing device. The composition containing thedye can be injected in a single dose into the bloodstream or injectedcontinuously to supply a continuous flow of the composition through thetarget site. The amount of the composition introduced can vary dependingon the target site and the length of time that the dye is to be excited.A light or energy source is continuously applied to the target site toexcite the dye and to cause the dye to fluoresce when released from theliposomes. An imaging device is used to capture the fluorescing lightfrom the dye to provide a visual indication that the dye is released.The release temperature of the liposomes are selected to release the dyeat a predetermined temperature so that when the dye is fluoresced andvisualized, the visualization provides the operator with an indicationthat the release temperature in the target site has been attained. Inone embodiment, the liposome composition is injected into the bloodstream so that the composition is able to provide a continuous supply ofthe dye for fluorescing during the hyperthermal treatment. In thismanner the operator is provided with a continuous indication that asufficient temperature is being maintained.

[0031] The method of the invention is primarily directed to a method ofheating tissue and cells in the tissue of an animal, particularly ahuman patient, at least to the temperature sufficient to kill or damagethe cells. Cell death or cell damage is known to occur when the tissuecells are heated to a temperature of about 5° C. above the normal bodytemperature of 37° C. Therefore, the method of the invention heats thecells in the tissue to a temperature of about 41° C., and preferably atleast 42° C. for a time sufficient to kill or damage the cells.Preferably, the heat source is applied to minimize unnecessary damage tothe surrounding cells and tissue.

[0032] In one embodiment of the invention, the tissue is heated to atemperature of at least 41° C. and preferably in the range of at leastabout 42° C. to about 50° C. Heating the tissue to at least 42° C.ensures that a sufficient temperature is obtained to thermally treat thetissue and the cells effectively. Preferably, the tissue is heated to atemperature below the protein denaturization temperature of the tissue.Protein denaturization begins to occur at about 50° C. to 51° C. andoccurs rapidly at temperatures of about 60° C. Preferably, the tissue isheated to a temperature of less than 60° C. and more preferably to atemperature of about 50° C. or less.

[0033] In one preferred embodiment, the tissue and the cells are heatedto a temperature of about 47° C. to about 49° C. for a time sufficientto kill or damage the cells without protein denaturization. The lengthof time that the tissue is heated will depend on the location of thetarget site, the size and dimensions of the target site, the desireddepth of penetration of the heat and the desired extent of thermaltreatment or damage of the tissue and cells in the target site.Typically, the heat source is applied for several minutes. In oneembodiment, the heat source is applied for about 1 to 15 minutes, andtypically about 5 to 10 minutes.

[0034] The heat source can be applied to a variety of the areas in thebody where the hyperthermal treatment is desired. The target site can betumors, organs, muscles and soft tissue. Examples of a target siteinclude blood vessels and arteries, esophagus and eyes. In oneembodiment, the method is suitable for hyperthermally treating theepithelial cells on the lens of the eye after cataract surgery. Othertarget sites include the retina and the choroid.

[0035] The target site is heated to the desired temperature tohyperthermally treat the target site using standard heating instrumentsand equipment for heating tissue and standard equipment for visualizingthe dye in the target site that has been released from the heatsensitive particles. For example, the heating equipment preferablyincludes suitable heat or energy source that is able to focus the heator energy on the target and is able to control heat and temperature ofthe tissue. The heat source can be an electrical resistance heatingelement, or an indirectly heated element. The heating device can alsohave an energy source for producing heat at the target site, such as aradio frequency (RF) device, ultrasonic generators, laser, or infrareddevice. One example of an RF generator device for hyperthermallytreating tissue in a selected target site is disclosed in U.S. Pat. No.6,197,022, which is hereby incorporated by reference in its entirety.Examples of suitable ultrasonic devices for delivering ultrasonichyperthermia are disclosed in U.S. Pat. Nos. 4,620,546, 4,658,828 and4,586,512, the disclosures of which are hereby incorporated by referencein their entirety.

[0036] In one embodiment, the heat source includes a probe having a tipwith the heating element or energy emitting element attached thereto.The energy emitting element can be an optical fiber operativelyconnected to a laser, infrared or ultraviolet light source. The probepreferably includes a suitable control mechanism for manipulating theprobe to the target site and a control for controlling the energyapplied to the target site.

[0037] A suitable device for hyperthermally treating the tissue in atarget site is shown in the FIGURE. The device 10 includes a probe 12having an optical fiber 14 with a distal end 16 for emitting a laserlight to heat the tissue 17. Preferably, the end 16 of optical fiber 14can focus the light source on the target site 17. Optical fiber 14 isconnected to laser generator 18 that is able to generate a laser beam ofsufficient intensity and within wavelength for hyperthermally treatingtissue to a temperature of at least 41° C. and preferably at least 42°C.

[0038] In a preferred embodiment, probe 12 includes a second opticalfiber 20 having a distal end 22 and a third optical fiber 24 having adistal end 26. Optical fiber 20 is operatively connected to a lightsource 28, such as a laser, that is able to emit a light beam having awavelength capable of fluorescing a fluorescent dye in the target areawhen the dye is released from the heat sensitive particles. Opticalfiber 24 is operatively connected to a suitable imaging device 30 forcapturing the fluoresced light from the excited dye and visualizing andproducing an image of the fluorescing dye in the target site. Imagingdevice 30 can be a CCD or a device equivalent to a funduscope. Anexample of a suitable funduscope is disclosed in U.S. Pat. No. 4,891,043to Zeimer, which is hereby incorporated by reference in its entirety.

[0039] In another embodiment of the invention, the probe can include aheating element or a device for receiving a heated fluid that cantransfer the heat to the tissue in the target site. The probe caninclude an expandable bladder member for receiving a heated fluiddelivered from a fluid-heating source. In still another embodiment, theexpandable bladder includes a permeable portion so that the heated fluidcan be applied directly to the target site. A suitable aspirating deviceis preferably included to remove the excess heating fluid when applieddirectly to the target site.

[0040] In one embodiment, the target site is the retina or choroid inthe eye of the patient. The heating and visualizing instrument includesa laser capable of focusing a laser beam on the target site where thelaser beam has a wavelength and intensity to heat the cells to atemperature of at least 42° C. In one embodiment, the laser heats thecells to a temperature of 50° C. or below and preferably to about 42° C.to 50° C. The instrument also includes or is used in combination with afunduscope to excite or fluoresce the dye that has been released in thetarget site to capture and visualize the fluorescing dye. A funduscopethat can be used is disclosed in U.S. Pat. No. 6,248,727, which ishereby incorporated by reference in its entirety. The laser source isselected to provide sufficient energy to heat the tissue in the targetsite to the desired temperature.

[0041] The fluorescent dye is encapsulated in a suitable heat sensitiveparticle and introduced into the patient in a location to be visualizedin the target site. The heat sensitive particles can be microcapsules,or nanocapsules that are able to release the dye at a temperature ofabout 41° C., and preferably 42° C. or higher. In preferred embodiments,the fluorescent dyes are incorporated into heat sensitive liposomes thathave a phase transition temperature at the temperature of hyperthermia.In one embodiment, the liposomes have a phase transition temperaturewithin the desired temperature range that tissue or cells are to beheated.

[0042] In one embodiment, the liposomes have a phase transitiontemperature of at least 41° C. and preferably at least 42° C. In apreferred embodiment, the liposomes have a phase transition temperatureof about 45° C. to about 50° C.

[0043] The liposomes can be made by various processes as known in theart. The phase transition temperature of the phospholipid is selected tocontrol the temperature that the dye and other components are releasedfrom the liposomes. Phospholipids are known to have different phasetransition temperatures and can be used to produce liposomes havingrelease temperatures corresponding to the phase transiture of thephospholipids. Suitable phospholipids include, for example,dimyristoylphosphatidyl choline having a phase transition temperature of23.9° C., palmitoylmyristoylphosphatidyl choline having a phasetransition temperature of 27.2° C., myristolypalmitoylphosphatidylcholine having a phase transition temperature of 35.3° C.,dipalmitoylphosphatidyl choline having a phase transition temperature of41.4° C. stearoylpalmitoylphosphatidyl choline having a phase transitiontemperature of 44.0° C., palmitoylstearolyphosphatidyl choline having aphase transition of 47.4° C., and distearolyphosphatidyl choline havinga phase transition temperature of 54.9° C. Another suitable phospholipidis a synthetic C₁₇ phosphatidyl choline from Aventi Inc. having a phasetransition temperature of about 48° C.-49° C.

[0044] The phase transition temperature and the release temperature ofthe liposomes can be selected by combining the different phospholipidsduring the production of the liposomes according to the respective phasetransition temperature. The phase transition of the resulting liposomemembrane is generally proportional to the ratio by weight of theindividual phospholipids. Thus, the composition of the phospholipids areselected based on the respective phase transition temperature so thatthe phase transition temperature of the liposome membrane will fallwithin the selected range. By adjusting the phase transition temperatureof the liposome membrane to the selected range, the temperature at whichthe liposomes release the dyes and other components can be controlledduring hyperthermia.

[0045] The liposomes in one embodiment of the invention are preferablyprepared so that the liposome membrane has a phase transitiontemperature of at least 42° C., and preferably about 42° C. to about 50°C. In a preferred embodiment, the liposomes leak or rupture at atemperature of about 49° C. or less, and typically between about 45° C.and 49° C. In one embodiment, the phospholipids have saturated acylgroups. For example, glycerophospholipids can be used that have two acylgroups having 8 or more carbon atoms and where at least one of the acylgroups have at least 10 carbon atoms and typically 12-18 carbon atoms.Examples of suitable phospholipids include hydrogenated lecithin fromplants and animals, such as egg yolk lecithin and soybean lecithin. Thephospholipid can also be phosphatidyl choline produced from partial orcomplete synthesis containing mixed acyl groups of lauryl, myristoyl,palmitoyl and stearoyl.

[0046] The liposomes can be prepared from a mixture ofdipalmitoylphosphatidyl choline and disteroylphosphatidyl choline in aweight ratio of 95:5 to about 5:95 and generally about 80:20 to about20:80. In one embodiment, the liposomes are made from a mixture ofdipalmitoylphosphatidyl choline and disteroylphosphatidyl choline in aratio of 45:55 to about 55:45 provide a phase transition temperature ofabout 46° C. to about 49° C.

[0047] The liposomes of the invention can be prepared by standardprocesses as known in the art. The liposomes can be unilamellar ormultilamellar and have a particle suitable for delivering the dye to thetarget site. In one embodiment, the liposomes have a particle size of asufficiently small size to be introduced into the bloodstream of thepatient in a location near the target site to flow through the targetsite.

[0048] The liposomes can contain a suitable osmotic pressure controllingagent that is physiologically acceptable to the patient. Examplesinclude sodium chloride, sugars such as glucose, mannitol and sorbitol,and amino acids such as glycine, aspartic acid and glutamic acid.Examples of suitable process for preparing liposomes are disclosed inU.S. Pat. No. 4,235,871 to Papahadjopoulos et al. and U.S. Pat. No.4,522,803 to Lenk, which are hereby incorporated by reference in theirentirety.

[0049] The liposomes of the invention contain a dye that is able tofluoresce and that can be visualized in the target site by exciting witha light source that is amenable to the target site and the patient. Thefluorescent dye can be any fluorescent that is suitable forencapsulation and is physiologically compatible. Preferably, thefluorescent dye is quenched when encapsulated at an appropriateconcentration. The quenching concentration is a sufficiently highconcentration to mask or minimize detection of fluorescence whenilluminated by a fluorescing light source. The quenching concentrationcan be determined by routine experimentation as known in the art. Whenheated, the heat sensitive liposomes rupture or leak the dye and othercomponents of the liposome so that the dye is diluted in the target siteto a suitable concentration where the dye can be fluoresced andvisualized upon excitation by a suitable light source. Examples ofsuitable dyes include 6-carboxyfluorescein and its derivatives. Suitablefluorescent dyes can be excited by an emit light at wavelengths that arenot strongly absorbed by the tissue. Other suitable dyes includeindocyanin green and aluminum phthalocyanine tetrasulfonate. It will beunderstood that the fluorescing light source and the visualizinginstrument are selected according to the wavelength of the fluorescingdye to visualize the dye.

[0050] In one embodiment, the dye is selected to fluoresce in thepresence of a light from an argon laser, a helium-neon laser or infraredlaser. Preferably the dye is selected to be compatible with the excitinglight or laser source to fluoresce when subjected to the light or laserbeam. A suitable dye is sold under the tradename D-275 by MolecularProbe, Inc. and fluoresces green when exposed to light from an argonlaser at 484 nm. A dye sold under the tradename D-1121 fluoresces orangewhen exposed to a long wavelength laser light at 560-574 nm. Onpreferred dye is an infrared excitable dye DiIc₁₈(7), which fluorescesat a wavelength of 740-780 nm.

[0051] In one embodiment of the invention, a fluorescent dye isencapsulated in liposomes having a phase transition temperature of 42°C. to 50° C., and preferably about 45° C. to 49° C. In anotherembodiment, the liposomes have a phase transition temperature to releasethe dye at a temperature of about 46° C. to about 49° C. The liposomesare injected into the bloodstream of the patient in a location where theliposomes flow to the target site. In some embodiments, the liposomescan be introduced directly to the target site intravenously,subcutaneously or topically. A hyperthermal heat source and a dyeexciting light source are applied to the target site. The hyperthermalheat source, which is preferably a laser light beam, is focused on thetarget site to heat the tissue and the cells to a temperature of atleast 42° C. to hyperthermally treat the tissue and kill the cells. Thehyperthermal heat source also heats the liposomes to a temperature atleast equal to the phase transition temperature to release the dye. Thefluorescing light source excites the dye so that the fluorescing dye inthe target site can be detected and visualized. By encapsulating thefluorescent dye in liposomes having a phase transition of at least 42°C., the detection of the fluorescing dye provides a positive indicationto the operator that the desired tissue temperature has been obtainedthat is necessary to hyperthermally treat the tissue. The phasetransition temperature of the liposomes is selected according to desiredminimum temperature that the tissue is to be heated. The hyperthermiaenergy source is applied to the target site for a time sufficient totreat the tissue to the desired level. Generally, the tissue is heatedto a temperature of at least 42° C. for 1-15 minutes and preferably 1-10minutes.

[0052] In one preferred embodiment of the invention, the liposomescontain a suitable drug or photosensitizing agent. The drugs preferablyshow a synergistic effect when combined with the hyperthermia treatmentof the invention. The release of the drugs from the liposomes provide animproved targeting effect by releasing the drugs by the heat source inthe target site. Suitable drugs include antitumor agents such ascisplatin, carboplatin, tetraplatin and iproplatin. Suitable anticancerdrugs include adriamycin, mitomycin C, actinomycin, ansamitocin and itsderivatives, bleomycin, Ara-C, daunomycin, metabolic antagonists such as5-FU, methotrexate, isobutyl5-fluoro-6-E-furfurylideneamino-xy-1,2,3,4,5,6hexahydro-2,4-dioxopyrimidine-5-carboxylate. Other antitumor agentsinclude melpharan, mitoxantrone and lymphokines. The amount of theparticular drug entrapped in the liposomes are selected according to thedesired therapeutic dose and the unit dose.

[0053] Examples of suitable photosensitive (photosensitizer) agentsinclude aminolevolunic acid, porphyrine derivatives, porpurinederivatives, NPE-6, ATX-10, plant derived photosensitizers. Othersynthetic sensitizers such as SNET₂ and Lutex can be used. Preferably,the photosensitizers are used in non-toxic amounts. In otherembodiments, the liposome compositions can contain liposomes thatencapsulate a hyperthermic potentiating agent such asperfluorooctyliodide, perfluorotributylamine, perfluorotripropylamine,and perfluorooctylbromide. Examples of liposome encapsulatedpotentiators are disclosed in U.S. Pat. No. 5,149,319 to Unger, which ishereby incorporated by reference in its entirety. Other bioactive agentsthat can be delivered to the target site by encapsulating in liposomesinclude anti-inflammatory agents, antibiotics, antibacterial agents,antifungal agents, anti-neoplastic agents and antiparasitic agents.Examples of anti-neoplastic agents include aclacinomycins, chromycinsand olivomycins.

[0054] In another embodiment of the invention, the liposome compositioncontains a mixture of liposomes having different phase transitiontemperature to release the components at different temperatures. In oneembodiment, the liposome composition contains liposomes encapsulating afirst dye and having a phase transition temperature of 42° C. to about45° C. and liposomes encapsulating a second dye and having a phasetransition temperature of about 50° C. or higher. In one embodiment, thesecond dye is encapsulated in liposome that release the dye at atemperature range of 50° C. to 60° C. Preferably, the second dye is ableto fluoresce at different color than the first dye so that the dyes aredistinguishable. In this embodiment, the liposome composition isdelivered to the target and the target site is subjected to hyperthermiatemperatures. As the tissue in the target site is heated to at least 42°C., the first liposomes rupture or release the first dye so that thefirst dye is visualized and detected in the target site. The detectionof the first dye in the target site enables the operator to monitor thetemperature of the tissue in the target site and to indicate that ahyperthermal temperature has been attained in the tissue at the targetsite. During hyperthermia, it is difficult to determine and monitor theactual temperature of the tissue and care must be taken to avoidoverheating of the tissue and denaturization of the proteins. Inpreferred embodiments of the invention, the hyperthermal treatment doesnot exceed the protein denaturization temperature. In this embodiment,the second liposomes are selected to rupture or release the second dyeat or slightly below the protein denaturization temperature. In thismanner, the second dye is released and visualized to provide theoperator with an indication that the tissue is heated to the proteindenaturization temperature. The heat source is then adjusted by theoperator to reduce the energy applied to the target site to avoidprotein denaturization.

[0055] In another embodiment, the liposome composition can containseveral liposomes that can leak or rupture at different temperatures torelease the dyes at incremental temperatures as the temperature of thetarget site increases. In one embodiment, the liposomes can be selectedto leak or rupture the dye at 2° C. intervals between about 42° C. and50° C. The dyes for each liposome can be different to fluoresce adifferent color so that the different colors indicate a differenttemperature of the target site.

[0056] In other embodiments of the invention, the tissue in the targetsite can be irradiated by beta radiation from strontium or iridiumisotopes. Gamma radiation from P³², iodine-95, and palladium-90 can alsobe used. The radioactive isotopes can be delivered as small particles tothe target site in combination with the hyperthermia treatment.

[0057] Although several embodiments have been chosen to illustrate theinvention, those skilled in the art will readily appreciate that variouschanges and modifications can be made without departing from the scopeof the invention.

What is claimed is:
 1. A method of hyperthermally treating tissue in ananimal, said method comprising the steps of: introducing a temperatureindicating substance into the bloodstream of said animal to flow througha target site, said temperature indicating substance including afluorescent dye encapsulated within a heat sensitive liposome, saidfluorescent dye being releasable from said liposome at a temperature ofat least 41° C., and applying a heat source to said target site andhyperthermally heating said target to at least 41° C. to release saiddye and to hyperthermally treat said target site for a time sufficientto kill cells in said tissue, and fluorescing and visualizing said dye.2. The method of claim 1, wherein said fluorescent dye is releasablefrom said liposome at a temperature of at least 42° C.
 3. The method ofclaim 1, wherein said fluorescent dye is releasable from said liposomeat a temperature sufficient to kill cells in said tissue substantiallywithout denaturing proteins in said tissue.
 4. The method of claim 1,wherein said liposome encapsulates a bioactive compound, and said methodcomprises heating said liposome to release said bioactive compound at atemperature of at least 42° C.
 5. The method of claim 4, wherein saidbioactive compound is heat activated at a temperature of at least 42° C.6. The method of claim 4, wherein said bioactive compound is anantiproliferative agent or an antitumor agent.
 7. The method of claim 4,wherein said bioactive agent is selected from the group consisting ofcisplatin, carboplatin, tetraplatin, iproplatin, adriamycin, mitomycinC, actinomycin, ansamitocin and bleomycin.
 8. The method of claim 1,wherein said heat source is a laser source, a microwave source, aninfrared source, or an ultrasonic source.
 9. The method of claim 1,wherein said heat source is a heated fluid source, and where said methodcomprises applying said heated fluid to said target site.
 10. A methodof detecting a threshold temperature and of hyperthermally treatingtissue in an animal, said method comprising the steps of: introducing afirst fluorescent dye encapsulated in a first heat sensitive liposomeinto the bloodstream of an animal in a location to flow through a targetsite in said animal, said first fluorescent dye being releasable fromsaid first heat sensitive liposome at a temperature of at least 41° C.,heating said target site to a temperature to release said firstfluorescent dye and fluorescing said first fluorescent dye to indicateand visualize a tissue temperature of at least 41° C., and continuingheating said target site at a temperature of at least 41° C. for a timesufficient to hyperthermally treat said tissue.
 11. The method of claim10, wherein said first fluorescent dye is releasable from said firstliposome at a temperature of at least 42° C. and said target site isheated at least to 42° C.
 12. The method of claim 10, comprising heatingsaid tissue to a temperature sufficient to kill cells in said tissue andbelow a protein denaturing temperature.
 13. The method of claim 10,comprising heating said target site to a temperature of at least about42° C. to about 50° C. for bout 1-10 minutes.
 14. The method of claim10, wherein said first liposome encapsulates a bioactive compound, andwherein said method comprises heating said first liposome to releasesaid bioactive compound at a temperature of at least 42° C.
 15. Themethod of claim 14, wherein said bioactive compound is heat activated ata temperature of at least 42° C.
 16. The method of claim 14, whereinsaid bioactive compound is an antiproliferative agent or an antitumoragent.
 17. The method of claim 14, wherein said bioactive agent isselected from the group consisting of cisplatin, carboplatin,tetraplatin, iproplatin, adriamycin, mitomycin C, actinomycin,ansamitocin and bleomycin.
 18. The method of claim 10, wherein said heatsource is a laser source, a microwave source, an infrared source or anultrasonic source.
 19. The method of claim 10, wherein said heat sourceis a source of heated fluid and said method comprises applying saidheated fluid to said target site.
 20. The method of claim 10, furthercomprising the step of introducing a second fluorescent dye encapsulatedin a second heat sensitive liposome into said bloodstream of saidanimal, said second fluorescent dye being releasable from said secondliposome at a temperature of at least 50° C., visualizing and detectingsaid second fluorescent dye released from said second liposomes andreducing said temperature of said tissue in response to said detectedsecond dye.
 21. The method of claim 20, wherein said second fluorescentdye is released from said second liposome at a temperature where proteindenaturization occurs, and wherein said temperature of said tissue isreduced below the protein denaturization temperature in response to saiddetected second fluorescent dye.
 22. The method of claim 20, comprisingheating said tissue in said target site to a temperature below a proteindenaturization temperature of said tissue and below said releasetemperature of said second fluorescent dye.
 23. A method ofhyperthermally treating tissue of an animal, said method comprising thesteps of: introducing a temperature indicating substance into thebloodstream of said animal to flow through a target site, saidtemperature indicating substance including a first fluorescent dyeencapsulated in a first temperature sensitive liposome, said firstfluorescent dye being releasable from said first liposome by heating toa temperature of at least 42° C., and a second fluorescent dyeencapsulated in a second temperature sensitive liposome, said secondfluorescent dye being releasable from said second liposome by heating toa temperature of at least 50° C., heating said target site and saidfirst temperature sensitive liposome to a temperature of at least 42°C., and fluorescing said first fluorescent dye to indicate an effectivetemperature for hyperthermally treating said tissue without releasingsaid second fluorescent dye from said second liposomes.
 24. The methodof claim 23, comprising detecting a fluorescence of said secondfluorescent dye and reducing said temperature of said tissue below aprotein denaturing temperature of said tissue.
 25. The method of claim23, wherein said first fluorescent dye fluoresces a color different froma color of said second fluorescent dye.
 26. The method of claim 23,wherein said first liposome comprises a phospholipid selected from thegroup consisting of dipalmitoylphosphatidyl-choline,dipalmitoylpyhosphatidyl-glycerol, and mixtures thereof.
 27. The methodof claim 23, wherein said second liposome comprises aC₁₇-phosphatidyl-choline, wherein said second liposome releases saidsecond fluorescent dye at a temperature of about 48° C.
 28. The methodof claim 23, wherein said first liposomes encapsulate a bioactivecompound.
 29. The method of claim 28, wherein said bioactive compound isselected from the group consisting of anti-proliferative agents andanti-tumor agents.
 30. The method of claim 28, wherein said bioactivecompound is cis-platin.
 31. The method of claim 28, wherein saidbioactive compound is a photoactivated compound, and wherein said methodcomprises activating said photoactivated compound to kill or inhibitmultiplication of cells in said target site.
 32. The method of claim 23,wherein said first temperature sensitive liposome leaks or ruptures at atemperature of about 42° C. to 50° C.
 33. The method of claim 23,wherein said first temperature sensitive liposomes leak or rupture at atemperature of about 45° C. to about 49° C.
 34. The method of claim 23,wherein said second temperature sensitive liposomes leak or rupture at atemperature of about 50° C. to 60° C.